The present invention is generally directed to novel glycosylated steroid derivatives for facilitating the transport of molecules across biological membranes and the blood-brain barrier. The invention is further directed to a novel glycosylation process for the efficient synthesis of these glycosylated steroid derivatives. To elicit the desired biological response, a molecule of diagnostic, prophylactic, or therapeutic interest [termed herein "therapeutically-significant molecule"or "therapeutically-significant-compound"]must be available in an effective concentration at its site of action. Many factors determine the concentration of a therapeutically-significant-compound that ultimately reaches the site of action, including the amount administered, and the extent and rate of the compound's absorption, distribution, biotransformation, and excretion. [Goodman and Gilman, The Pharmacological Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., Inc., New York, 1980, pp. 1-39]. The foregoing factors may, in turn, be influenced by the route chosen for administration of the therapeutically-significant-compound.
The most common routes of administration of therapeutically-significant-compounds are parenteral (intravenous, subcutaneous, and intramuscular) and enteral (oral ingestion), although methods to administer therapeutically-significant-compounds across the skin or mucosa (oral, nasal, rectal, vaginal, etc.) also are known. Parenteral methods are considered to be extremely effective in general, allowing for rapid increases in blood levels of a wide range of therapeutically-significant-compounds. Parenteral methods are advantageous in that they circumvent first-passage hepatic metabolism. However, parenteral administration of a therapeutically-significant-compound can cause pain, irritation, possible tissue damage over the long term, and carries a potential risk of infection. In addition, parenteral methods frequently are inconvenient, particularly those that are restricted to trained medical personnel (e.g., intravenous methods).
Enteral methods are more convenient than parenteral methods, and generally are more economical and acceptable to the recipients. However, orally administered, therapeutically-significant-compounds may be inefficiently absorbed and the time from ingestion to absorption may prohibit effective use in emergency situations. Moreover, many therapeutically-significant-compounds cannot be orally administered as they are destroyed, prior to reaching their site of action, by the digestive enzymes, acid, and surface-active lipids in the gut. Other therapeutically-significant-compounds are subject to extensive, first-passage hepatic metabolism, rendering them ineffective following oral administration.
Non-parenteral methods which circumvent problems associated with instability of drug preparations in the gut and first-passage hepatic metabolism long have been sought. Administration via transdermal, oral mucosal, rectal, and nasal routes are among the alternatives which have been explored. Such alternatives further include administering the therapeutically-significant-compound orally, but encapsulated in a protective delivery system designed to extrude the contents at a predetermined point in the lower gastrointestinal tract. However, the efficacy of these alternative drug delivery methods often is limited by poor absorption of the therapeutically-significant-compounds at the site of delivery or application. Effective strategies to enhance absorption of therapeutically-significant-molecules across cell membranes could enhance the efficacy of many known drug preparations which are poorly absorbed regardless of the method of administration. Such strategies to enhance transmembrane absorption could be particularly useful for therapeutically-significant-compounds that are administered across the skin and mucosal tissues, including mucosal tissues of the gastrointestinal, genitourinary, and respiratory tracts.
The basic structural unit of biological membranes is a phospholipid bilayer, in which are embedded proteins of various size and composition. The surfaces of the phospholipid bilayer, which project into the aqueous cellular environment, are formed by the hydrophilic heads of the phospholipids; the interior, by the fatty acyl hydrophobic tails. The membrane proteins may be involved in transport processes and also may serve as receptors in cellular regulatory mechanisms.
Natural mechanisms for traversal of biological membranes include passive diffusion, facilitated diffusion, active transport, receptor-mediated endocytosis and pinocytosis. Passive diffusion works best for small molecules which are lipid-soluble. However, biological membranes are essentially impermeable to most water-soluble molecules, such as nucleosides, amino acids, proteins, and other hydrophilic, therapeutically-significant-molecules. Such molecules enter cells via some type of carrier-mediated transport system in which specific entities facilitate traversal of the membrane. Natural carriers for facilitating traversal of the membrane are of limited utility, however, as such carriers will accept substrates of only a predetermined molecular configuration. Many therapeutically-significant-compounds are not efficiently absorbed because they are neither lipophilic enough to cross cell membranes by passive diffusion nor recognized by the natural transport systems.
Strategies to enhance the uptake of therapeutically-significant-molecules across biological membranes have been investigated previously and fall into two broad categories. The first category includes all strategies in which the structure of the therapeutically-significant-compound is changed, either by making the compound more lipophilic itself, or by conjugating the compound to other entities known to interact with phospholipid membranes. The common goal of these strategies has been to increase passive diffusion across the membrane by lowering the energy barrier to diffusion and/or by increasing the local concentration of the compound at the membrane surface. Also included in the first category is a strategy for conjugating the therapeutically-significant-compound to entities known to interact with transport machinery embedded in the biological membranes, the goal being to take advantage of the transport machinery (either active or facilitated transport or receptor-mediated endocytosis) to increase delivery of the compound across the membrane.
Many investigators are studying the feasibility of increasing the efficacy of hydrophilic compounds by conjugating these compounds to entities known to interact with phospholipid membranes. Among the techniques reported are utilization of oligonucleotide-cholesterol conjugates [Letsinger RL et al. "Cholesteryl-conjugated oligonucleotides: Synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture." Proc. Natl. Acad. Sci. USA 86: 6553-6556 (September 1989); Stein CA et al. "Mode of Action of 5'-Linked Cholesteryl Phosphorothioate Oligodeoxynucleotides in Inhibiting Syncytia Formation and Infection by HIV-1 and HIV-2 in Vitro." Biochemistry 30:2439-2444 (1991)].
Targeting molecules to the brain requires traversal of the blood-brain barrier--a capillary-including system, with unique morphological characteristics, which acts as a system-wide cellular membrane separating the brain interstitial space from the blood. Like biological membranes, the blood-brain barrier is relatively impermeable to many hydrophilic, therapeutically-significant-compounds. Among the strategies which have been developed for targeting compounds to the brain are direct delivery by invasive procedures, intra-arterial infusion of hypertonic substances, and conversion of hydrophilic compounds to lipid-soluble entities. Recent attempts at facilitated transport, as described in U.S. Pat. No. 4,902,505, involve coupling a hydrophilic peptide of interest to a peptide carrier which, by itself, is capable of traversing the barrier via receptor-mediated transcytosis.
The second category of strategies to enhance uptake includes those in which the therapeutically-significant-compounds are administered to specific body surfaces as admixtures with other molecules which are known to permeabilize membranes. For example, several investigators have attempted to mix insulin with adjuvants, such as bile salts, which might enhance nasal insulin absorption. [Hirai et al., Int. J. Pharmaceutics 9:165-184 (1981); Hirai et al. Diabetes 27: 296-199 (1978); British Patent No. 1,527,506; U.S. Pat. No. 4,153,689; and Pontiroli et al. Br. Med. J. 284:303-386 (1982)]. EP 0 444 778 describes the use of alkyl saccharides to enhance the penetration of topically applied drugs across mucus-covered epithelial tissues in general, and the corneal epithelium, in particular. U.S. Pat. No. 4,865,848 to Cheng et al., issued Sep. 12, 1989, discloses the use of sucrose esters, particularly sucrose monolaurate, for enhancing the transdermal flux of transdermally-delivered drugs. U.S. Pat. No. 4,746,508 to Carey et al, issued May 24, 1988, reports the use of fusidic acid and cephalosporin derivatives to increase the permeability of human and animal body surfaces to drugs.
The glycosylated steroid derivatives of the present invention are known to interact with phospholipid membranes, thereby enhancing the penetration of therapeutically-significant-compounds through such membranes, including biological membranes. Like some of the previously used adjuvants and enhancers (e.g., cholic acid and fusidic acid derivatives) the novel derivatives of the present invention are amphiphilic in a facial sense. However, the novel steroid derivatives of the present invention have significantly different structures in that they are glycosylated on their hydrophilic surfaces, a feature not shared by any of the previously-known, facially-amphiphilic steroids. The present inventors have discovered that glycosylation on the hydrophilic surfaces significantly changes both the solubility properties of the steroids and the manner in which they associate. Many of these glycosylated steroids have been shown by the inventors to be more effective that the parent, non-glycosylated, steroids, in permeabilizing both artificial and biological membranes. The novel glycosylated steroid derivatives of the present invention, therefore, may be used to increase the delivery of therapeutically-significant-compounds across cell membranes, either in admixture with the compounds or as conjugates to the compounds.
Prior to the present invention, no method existed for synthesizing all of the glycosylated steroid derivatives of the present invention. Many glycosylation reactions using thioglycosides have been reported. [Ferrier RJ et al. "A Potentially Versatile Synthesis of Glycosides." Carbohydrate Research 27: 55-61 (1973); Garegg PJ et al. "A reinvestigation of glycosidation reactions using 1-thioglycosides as glycosyl donors and thiophilic cations as promoters" Carbohydrate Research 116: 162-5 (1983); Nicolaou KC et al. "A Mild and General Method for the Synthesis of O-Glycosides." J Am Chem Soc 05:2430-2434 (1983); Lonn H. "Synthesis of a tri- and a hepta-saccharide which contain .alpha.-L-fucopyranosyl groups and are part of the complex type of carbohydrate moiety of glycoproteins."Carbohydrate Research 39:105-113 (1985); Andersson F et al. "Synthesis of 1,2-cis-linked glycosides using dimethyl(methylthio)sulfonium triflate as promoter and thioglycosides as glycosyl donors." Tetrahedron Letters pp. 3919-3922 (1986); Brown DS et al. "Preparation of cyclic ether acetals from 2-benzenesulphonyl derivatives: a new mild glycosidation procedure." Tetrahedron Letters 29/38: 4873-4876 (1988); Ito Yet al. "Benzeneselenenyl triflate as a promoter of thioglycosides: a new method for O-glycosylation using thioglycosides." Tetrahedron Letters pp. 1061-4 (1988); Dasgupta F. et al. "Alkyl sulfonyl triflate as activator in the thioglycoside-mediated formation of .beta.-glycosidic linkages during oligosaccharide synthesis." Carbohydrate Research 177: c13-c17 (1988)]. However, none of these reported methods teach the use of a glycosyl sulfoxide as a glycosylating agent.
Utilization of an activated glycosyl sulfoxide intermediate in a process for glycosylating steroids, previously has been reported by the inventors in J. Am. Chem. Soc. 111:6881-2 (1989), the content of which is hereby incorporated by reference. However, the reported method represents only preliminary results on the glycosylation of steroids of the Formula (I). More specifically, further experimentation in the series has revealed unique reaction conditions which are necessary to achieve the efficient and stereo-selective synthesis of glycosylated compounds of the Formula (I). The reaction solvent used plays a critical role in the stereoselectivity of glycosylation. Using a nonpolar, aprotic solvent increases selectivity for alpha (.alpha.) glycosidic bond formation while the use of a polar, aprotic solvent such as propionitrile increases selectivity for beta (.beta.) glycosidic bond formation. The type of sulfoxide used in the glycosylation reaction also affects the outcome of the reaction. For example, it is vital to use the para-methoxy phenyl sulfoxide as the leaving group in the novel process described herein to obtain good yields of beta (.beta.) selectivity in the glycosidic bond formation. The yield of the glycosylation reaction yielding alpha (.alpha.) or beta (.beta.) glycosidic linkages also may be increased by the use of less than one equivalent of triflic anhydride in the glycosylation process.
Finally, the protecting groups on the glycosyl donor also have an impact on the stereochemical course of the glycosylation reaction. When the protecting group used on the glycosyl donor is pivaloyl, only beta (.beta.) glycosidic bonds are formed in the glycosylation process, regardless of whether an aprotic, non-polar solvent or an aprotic, polar solvent is used for the reaction. The above factors taken together indicate that one skilled in the art could not have practiced the invention without the detailed further experimentation provided herein.